Nucleic Acids Research, Vol 26, Issue 20 4733-4738, Copyright © 1998 by Oxford University Press
ML Engel and DS Ray
The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia
fasciculata consists of minicircles and maxicircles topologically
interlocked in a single network per cell. Individual minicircles replicate
unidirectionally from either of two replication origins located 180 degrees
apart on the minicircle DNA. Initiation of minicircle leading-strand
synthesis involves the synthesis of an RNA primer which is removed in the
last stage of replication. We report here the purification to near
homogeneity of a structure-specific DNA endo-nuclease based on the RNase H
activity of the enzyme on a poly(rA).poly(dT) substrate. RNase H activity
gel analysis of whole cell and kinetoplast extracts shows that the enzyme
is enriched in kinetoplast fractions. The DNA endonuclease activity of the
enzyme is specific for DNA primers annealed to a template strand and
requires an unannealed 5' tail. The enzyme cleaves 3' of the first base
paired nucleotide releasing the intact tail. The purified enzyme migrates
as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 A and a
sedimentation coefficient of 3.7 s, indicating that the protein is a
monomer in solution with a native molecular mass of 32.4 kDa. These results
suggest that the enzyme may be involved in RNA primer removal during
minicircle replication.
ARTICLES
A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata
Molecular Biology Institute and Department of Molecular, Cell and Developmental Biology, 611 Circle Drive East, University of California Los Angeles, Los Angeles, CA 90095-1570, USA.
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